Mutational analysis of Hsp90α dimerization and subcellular localization: dimer disruption does not impede ‘in vivo’ interaction with estrogen receptor

The molecular chaperone Hsp90 has been found ubiquitously as a predominantly cytoplasmic dimer. By interacting with cytoplasmic or nuclear proteins such as pp60v-src or steroid receptors, Hsp90 helps its targets to become competent for full biological activity. Mutational deletion analysis of some properties of chicken Hsp90 alpha was undertaken after transient transfection of the constructs in COS7 cells. First, Hsp90 mutants were analyzed for their ability to behave as cytosolic dimers. We confirmed that the C-terminal Hsp90 region (amino acids 446-728) was sufficient for dimerization, and found that deletion of three small subregions in the 200 C-terminal residues precluded Hsp90 dimer formation. Moreover, we demonstrated that the N-terminal region of the protein (1-442) was not involved in dimerization. Second, the subcellular localization of the wild-type (WT) protein and mutants was analyzed by specific immunodetection and confocal microscopy. Most of the mutants were cytoplasmic like Hsp90WT, a nuclear localization being barely detectable in the WT protein or in mutants with a C-terminal truncation equal to or shorter than 286 residues. Surprisingly a mutant encoding the N-terminal region (1-285) was nuclear localized. In addition, the in vivo interaction between the cytoplasmic Hsp90 and the nuclear ER was documented after coexpression of both proteins in the same cells: some Hsp90 was shifted into the nucleus via its interaction with ER. From an analysis of dimeric or monomeric cytoplasmic Hsp90 mutants, we found that disruption of Hsp90 dimer did not systematically impede its interaction with ER. Finally, Hsp90WT and cytoplasmic mutants were tested for their ability to rescue from lethality a yeast strain deleted of both Hsp90 genes. Interestingly, the delta 661-677 mutant that showed an impaired dimerization but interacted with ER was able to confer viability, while the mutant deleted of the 30 C-terminal residues (NC6) was monomeric, did not confer viability and did not interact with ER. We therefore suggest that Hsp90 properties analyzed here are not necessarily interdependent.